Multiple Sclerosis (MS) is an immune-mediated CNS disease characterized by perivascular CD4 T cell and mononuclear cell inflammation and subsequent primary demyelination of axonal tracks leading to progressive paralysis. Although MS is generally considered to involve an autoimmune pathology, little is known regarding its etiology and there are limited therapeutic strategies available to specifically inhibit and prevent ongoing disease. As CD40/CD154 interactions are involved in multiple levels in the immune response (including IL-12-dependent Th1 differentiation, maturation of APC function and release of various inflammatory effector molecules), blockade of this interaction presents an attractive strategy for treating ongoing autoimmune disease. Indeed, our preliminary data clearly demonstrate that short-term therapy with anti-CD154 antibody is effective at blocking both the induction, and more importantly, the progression of ongoing PLP139-151-induced relapsing experimental autoimmune encephalomyelitis (R-EAE). We have also shown that anti-CD154-mediated disease inhibition is associated with a delay in differentiation of myelin-specific Th1 effector cells and inhibits the retention and possibly the expansion/recruitment of immune effector cells in the CNS. This project will elucidate the cellular and molecular mechanisms by which CD154 blockade prevents the functional activity of encephalitogenic effector Th1 cells in acute and relapsing EAE and thus inhibits expression of clinical R-EAE. The hypothesis under test is that short-term therapeutic CD154 blockade transiently prevents CD4+ T cell effector function through prevention of cell expansion in and/or recruitment of immune cells to the CNS target organ and through blockade of a critical pro-encephalitogenic signal(s) provided to T cells during their development in the periphery. Aim 1 will examine the effects of CD154 blockade on T cell entry, expansion and effector function within the CNS of mice with PLP139-151- induced R-EAE and the effects on production of chemokines and cytokines required for recruitment of bystander mononuclear cells to the CNS. Aim 2 will investigate the cellular and molecular events affected by CD154 blockade on development and differentiation of T cells specific for both the disease-initiating T cells and T cells specific for endogenous epitopes released during the process of epitope spreading. Aim 3 will analyze in the effects of CD154 blockade on dendritic cell differentiation and function during ongoing immune responses both in vivo and in vitro. These studies should further our understanding of the role of CD40/CD154 interactions in regulating induction and progression of CNS autoimmunity and provide vital information relative to optimizing strategies for the blockade of this interaction for the treatment of human autoimmune diseases.